PTEN is a tumor suppressor that is mutated in many forms of human cancer. PTEN encodes a phosphatase that recognizes the important second messenger, phosphatidylinositol-3,4,5-triphosphate (Pl- 3,4,5-P), and removes the 3'-phosphate from the inositol ring. PTEN therefore antagonizes PI-3 kinase, which phosphorylates the inositol ring at the same position. PI-3 kinase is an oncogene in its own right that is frequently mutated and amplified in tumors. Although PI-3 kinase and PTEN potentially influence many signaling pathways, perhaps the best understood pathway involving these genes is the insulin/IGF pathway, which is conserved in homo sapiens, c. elegans and drosophila. In these species, PTEN acts downstream of the insulin/IGF receptor, the insulin receptor substrate (IRS) adaptor and PI-3 kinase and upstream of AKT kinase. The most significant targets of AKT are the FOXO transcription factor genes. Activation of the PI-3 kinase pathway is highly oncogenic;however, it remains unclear how many components of the pathway contribute to oncogenesis. This application will focus on three fundamental problems in understanding how the PI-3 kinase pathway drives tumor formation. In aim 1, we will explore the possibility that pathological over expression of IRS2 in tumors can stimulate PI-3 kinase and collaborate with other alterations on the Pl- 3 kinase pathway to boost the PI-3 kinase signal. We will introduce IRS2 into normal human cells and examine them for alterations in growth, differentiation, and signaling. The minimal domain that is competent for inducing signaling and altered growth will be defined. We will measure the frequency of over expression in tumors and measure the effect of RNAi on tumor cell line growth and signaling. Lastly, we will establish a transgenic model of IRS2 overexpression to test the hypothesis that it is an oncogene . In aim 2, we will determine the effect of PTEN dose on tumor formation in mice. Loss of one copy of PTEN and reduced PTEN protein expression are extremely common in human malignancy. We therefore will reduce expression of PTEN in mice using RNAi and examine its effect on tumor formation. In aim 3, we will investigate how PTEN regulates FOXO transcription factors to activate a target gene. DNA elements will be identified on the promoter that coordinate gene activation. A combination of overexpression, RNAi, and chromatin immuno- precipitation experiments are planned to identify the mechanism of activation.